Invasive anal cancer (IAC) is a health crisis for gay, bisexual, transgender & other men who have sex with men (MSM), where risk for disease is now 20-40-fold higher than all U.S. males.9-12 Thirteen human papillomaviruses (high-risk HPVs) cause most invasive cervical cancers (ICC) in women & likely cause most IACs.13 High-risk HPV infections are sexually transmitted between partners. Persistent infections, together with their associated high-grade dysplasias, strongly predict ICCs.14-17 Recent data suggests we poorly understand HPV infections in men, especially among MSM who are at highest risk for IAC.18-25 Cervical cytology using Papanicolaou's staining (Pap test) significantly reduced ICC beginning in the mid-1950's; & cytology specimens are currently collected using cytobrush, a tool poorly adapted to anal sampling.26,27 Experts now recommend anal Pap test for MSM every 1-2 years, using Dacron swab passed blindly through the anal verge.28 Dacron-cytology specimens are marginally sufficient & require diagnostic follow-up for any detected abnormalities, a lower threshold than used for cervical cytology. Our pilot data show that sensitivity & specificity of anal cytology to predict HG-AIN is improved 1.5-fold using nylon-flocked swab, but only improved specificity 1.3-fold to 73%. Although most IACs test positive for HPV using PCR, the high prevalence of infection among MSM without cancer makes HPV PCR genotyping a poorly specific screening test, with low positive predictive value (PPV). High-threshold, nucleic acid HPV assays (molecular HPV tests) are calibrated to better predict high-grade cervical dysplasia in older females without atypias & to triage women to colposcopy with atypical squamous cells on cytology; they are not calibrated for IAC screening. Two molecular HPV tests that detect viral DNA & -mRNA may be relevant for IAC screening: HPV-Hybrid-capture II (HC-2) & -APTIMA. Both tests detect the 13 highest-risk HPVs; APTIMA detects HPV66, additionally. HC-2 detects HPV-DNA >1 pg/mL.29 HPV E6/E7 are often detected at higher levels where HPV is integrated into human DNA, a hallmark of cancer.30 Molecular HPV tests significantly reduce diagnostic follow-up referrals for women with equivocal cytology, limiting costly & invasive procedures, & while these tests improve detection of in situ & ICCs, they have not been explored as adjunctive tests for IAC screening. Also, sufficient attention has not been paid to improving the quality of anal Pap test specimens. This study seeks to evaluate two Pap test collection protocols & molecular HPV tests, as biomarker assays, using specimens collected at a single examination visit & randomized controlled study design. Optimizing the sequence of biomarker assays & cytology to predict HG- AIN will decrease morbidity & mortality, & lower use of costly & invasive diagnostic testing. We will evaluate the contribution that molecular HPV testing makes when simultaneously or sequentially positive tests, with or without (anal) cytology, are used to predict HG-AIN. Sensitivity, specificity, & PPV for anal cancer screening algorithms & their cost-effectiveness to prevent invasive anal cancer will be evaluated to inform practice.